You'd Prefer An Argonaute

Role of a ribosome-associated E3 ubiquitin ligase in protein quality control

Mario H. Bengtson & Claudio A. P. Joazeiro

Nature 467, 470–473, 23 September 2010.
doi:10.1038/nature09371

This week’s insightful summary and analysis by David Weinberg:

In their 2010 Nature paper, Bengtson & Joazeiro demonstrate that proteins being translated from non-stop mRNAs are targeted to the proteasome by the E3 ubiquitin ligase Ltn1.  A non-stop mRNA is defined as any mRNA that lacks a stop codon that is recognized by the ribosome.  Natural causes of non-stop mRNAs may include mutations in termination codons (at the DNA level or due to transcription errors), readthrough of bona fide termination codons, premature or alternative cleavage and polyadenylation within coding regions, or the initiation of 3′-5′ mRNA decay on messages being translated.  Because a non-stop mRNA can only be recognized as such after at least one round of translation, non-stop protein products are necessarily generated from non-stop mRNAs.  The translation of non-stop mRNAs is problematic for the cell because it results in the production of aberrant proteins and, perhaps more importantly, sequesters ribosomes as a result of the failure to recruit release factors.  While the quality control pathway that recognizes non-stop mRNAs in eukaryotes has come into focus over the past decade, the details of how non-stop protein products are recognized and degraded has been relatively under-studied.  Here, the authors synthesize previously-published results with their own observations to provide the first comprehensive picture of the non-stop protein decay pathway in eukaryotes.

The story began in 2009 when Joazeiro’s lab identified the E3 ubiquitin ligase Listerin in a forward genetics screen for neurodegeneration in mice.  Seeking to gain insight into the cellular function of Listerin, the authors turned to its homolog Ltn1 in the budding yeast S. cerevisiae.  Since Ltn1 had been previously pulled out in a yeast genetic screen for non-stop decay genes, Bengtson & Joazeiro go after the precise role of Ltn1 in this relatively-uncharacterized pathway.  After verifying previously-published results that convincingly demonstrate a role for Ltn1 in the quality control of non-stop proteins, the authors go one step further and implicate its ability to specifically bind to and ubiquitinate non-stop proteins as an essential part of this pathway.

Although the E2-binding RING domain in Ltn1 is shown to be required for its function in non-stop protein decay, the identity of the E2 binding partner is not addressed here.  A traditional pulse-chase experiment is used to show that Ltn1 promotes the turnover of newly-synthesized non-stop proteins, which raises the question of how non-stop proteins are recognized as aberrant and thereby targeted for degradation.  A hint (or perhaps even the answer) came from previously-published observations that hard-coding 12 Lys residues in an otherwise-normal protein causes instability and that long tracts of Lys or Arg cause translational arrest (presumably due to electrostatic interactions between the nascent peptide and the ribosome exit tunnel).  If a ribosome were to translate through the 3′-UTR of a non-stop mRNA and reach the poly-A tail, translation through the poly-A tail would naturally generate a C-terminal poly-Lys tract in the protein product that might similarly stall translation.  Indeed, the authors show that hard-coding Lys residues recruits Ltn1 and leads to ubiquitination.  Intriguingly, products associated with apparently-stalled ribosomes are specifically targeted to Ltn1, while the protein products from ribosomes that efficiently translate through the poly-K tract are not.  This suggests that the translational stall, rather than the poly-Lys tract, is the signal for Ltn1 recruitment.  Unfortunately the authors don’t address perhaps the most interesting follow-up question here:  Does any translational stall (e.g., one caused by stretches of rare codons) trigger Ltn1-dependent ubiquitination, or is it somehow specific for non-stop proteins?  Aside from identifying the poly-K tract as sufficient for Ltn1 recruitment, no additional insight is provided into how this is accomplished at the molecular level.  Additional experiments show that the nascent non-stop protein is associated with ribosomes and, moreover, that Ltn1 itself is predominantly associated with ribosomes.

The paper concludes with an attempt to demonstrate the biological relevance of Ltn1 by identifying a phenotype in the ltn1 knock-out strain.  While the strain shows no growth defect in standard media, the addition of either an antibiotic or nonsense suppressor mutation – both which would facilitate stop codon readthrough – reveals a slow-growth phenotype for the knock-out strain.  Thus, the authors conclude that Ltn1 confers resistance to stress caused by the production of non-stop proteins, but it is unclear if the slow-growth phenotypes are due to the accumulation of the non-stop proteins themselves or the depletion of translation-competent ribosomes.

In my opinion, the most interesting aspect of the pathway characterized in this paper is how it compares to the analogous pathway used by prokaryotes.  The ssrA/tmRNA pathway in prokaryotes similarly depends on the tagging of stalled nascent polypeptides and their subsequent degradation by energy-dependent proteases.  However, in the case of prokaryotes – whose mRNA lack poly-A tails – the tagging sequence is provided in trans by a tmRNA molecule that recognizes ribosomes that have reached the end of an mRNA.  In contrast, eukaryotes appear to take advantage of the existing poly-A tail to accomplish a similar feat without the need for a trans-acting factor.  Interestingly, the tmRNA includes a stop codon that triggers translation termination of non-stop messages, while the eukaryotic pathway appears to never ‘officially’ terminate translation.  This key difference perhaps warrants further investigation, as it seems unlikely that eukaryotes would altogether bypass a requirement for translation termination to recycle ribosomes from non-stop mRNAs.

While many questions remain – including how this function for Ltn1 is related to the neurodegeneration phenotype observed in Listerin mutant mice – this paper provides a satisfying initial characterization of the eukaryotic non-stop protein decay pathway, albeit with the help of many previously-published results and limited novel insight.